Measurement of discrimination against 18O during dark respiration in plants is currently accepted as the only reliable method of estimating the partitioning of electrons between the cytochrome and alternative pathways. In this paper, we review the theory of the technique and its application to a gasphase system. We extend it to include sampling effects and show that the isotope discrimination factor, D, is calculated as -dln(1 + δ)/dlnO*, where δ is isotopic composition of the substrate oxygen and O* = [O2]/[N2] in a closed chamber containing tissue respiring in the dark. It is not necessary to integrate the expression but, if the integrated form is used, the resultant regression should not be constrained through the origin. This is important since any error in D will have significant effects on the estimation of the flux of electrons through the two pathways.